Human herpesvirus 8 (HHV-8) specifies four viral interferon regulatory factor homologues (vIRFs) that function to inhibit cellular IRFs in addition to other components of cellular defense pathways that promote cell cycle arrest and apoptosis in response to virus infection. Cellular proteins targeted for inhibition by vIRF-1 include p53, ATM, GRIM19, Smad transcription factors, and p300/CBP transcriptional co-activators required for IRF-?mediated responses. This laboratory has identified an entirely novel class of interaction, between vIRF-1 and stress-responsive, pro-apoptotic BH3-only proteins (BOPs) Bim and Bid, demonstrated to be inhibited by vIRF-1 binding, and other newly recognized BOP targets. Furthermore, we have identified partial localization of vIRF-1 to mitochondria, suggesting that vIRF-1 can inactivate BOPs directly at their site of action. vIRF-1- induced nuclear translocation of Bim represents a secondary, unique mode of inactivation. vIRF-1:BOP interaction is via the Bid-BH3B-related BOP-binding domain (BBD) of vIRF-1 (residues 170-187) and the BH3 domains of targeted BOPs. Both Bim and Bid are induced by HHV-8 lytic replication and Bim, at least, is a powerful inhibitor of virus production, suggesting that Bim and probably other BOPs need to be controlled effectively by HHV-8 for virus replication to occur. Indeed, vIRF-1 BBD-mediated interactions contribute specifically and significantly to promotion of HHV-8 productive replication and vIRF-1-mediated apoptotic inhibition. However, the precise contributions of each vIRF-1:BOP interaction and the significance of other vIRF-1:protein interactions in virus biology are at present unknown. Furthermore, our newly-identified mitochondrial localization of vIRF-1 has yet to be assessed functionally. This application proposes: identification of the molecular/specificity determinants of BOP/BH3 targeting and inhibition by vIRF-1 (Aim 1); molecular dissection and dissociation of other protein-binding and functional domains of vIRF-1 (Aim 2); establishing the contributions of each vIRF-1:protein interaction and mitochondrial-localized activity of vIRF-1 in the context of HHV-8 infection (Aim 3). The project comprises a comprehensive, yet focused analysis of an HHV-8 protein with a demonstrated positive role in virus replication and which provides a uniquely valuable tool to study the relative importance of multiple (vIRF-1-targeted) cellular defense pathways in virus biology. Information generated from this study could provide the basis for development of novel anti-viral therapies.